Myosin is found in all eukaryotic cells and appears to be involved in diverse cellular motile processes such as cytokinesis. There are at least two genes for vertebrate nonmuscle myosin heavy chains (MHCs), MHC-A and B. The nonmuscle MHC-A gene is expressed abundantly in fibroblasts, epithelial cells and lymphoid cells, but less abundantly in neuronal cells and differentiated muscle cells. We have been studying the mechanisms responsible for the cell type-dependent transcription for the nonmuscle MHC-A gene. We have previously identified three clustered cis-regulatory elements (A, C and F) in intron 1, which modulates transcription in a cell type-dependent manner (J. Biol. Chem. 273, 9168-9178, 1998). In this study, we identified putative trans-acting factors for element F which contains an E-box. Yeast one hybrid cloning shows that transcriptional factors TFEC, TFE3 and USF2 interact with the F element. All three proteins contain a basic helix loop helix and a lucine zipper region and TFEC and TFE3 especially show very high homology in these two regions. In vitro binding activities of TFEC and TFE3 have been analyzed by gel shift assays using in vitro transcribed and translated full-length cDNAs. TFEC and TFE3 can form homodimeric complexes with the F element. The binding activity of the TFEC homodimer is higher than that of the TFE3 homodimer. In addition to homodimers, TFEC and TFE3 can also form a heterodimer and bind to the F element.